A DNA-Based FLIM Reporter for Simultaneous Quantification of Lysosomal pH and Ca²⁺ during Autophagy Regulation

pH and Ca²⁺ play important roles in regulating lysosomal activity and lysosome-mediated physiological and pathological processes. However, effective methods for simultaneous determination of pH and Ca²⁺ is the bottleneck. Herein, a single DNA-based FLIM reporter was developed for real-time imaging and simultaneous quantification of pH and Ca²⁺ in lysosomes with high affinity, in which a specific probe for recognition of Ca²⁺ was assembled onto a DNA nanostructure together with pH-responsive and lysosome-targeted molecules. The developed DNA reporter showed excellent biocompatibility and long-term stability up to ∼56 h in lysosomes. Using this powerful tool, it was discovered that pH was closely related to Ca²⁺ concentration in lysosome, whereas autophagy can be regulated by lysosomal pH and Ca²⁺. Furthermore, Aβ-induced neuronal death resulted from autophagy abnormal through lysosomal pH and Ca²⁺ changes. In addition, lysosomal pH and Ca²⁺ were found to regulate the transformation of NSCs, resulting in Rapamycin-induced antiaging.